This invention concerns a novel class of bacteriolytic proteins capable of lysing certain gram negative bacteria, the processes for inducing and obtaining this class of proteins from insect hemolymph and the processes for their utilization.
Recent developments in biochemistry have made available recombinant bacteria that synthesize enzymes and other nonbacterial proteins. These genetically engineered bacteria differ from those that hitherto occurred naturally by containing, along with their own genes, at least portions of genes inserted from other organisms which have instructions encoded in their DNA for synthesizing proteins having important biomedical applications. When the animal gene is properly integrated into the bacterial genome, the resulting recombinant bacteria produces the protein specified by the animal gene. Cultures of the recombinant bacteria are easily grown at low cost and hold out the promise of efficiently producing important proteins. Examples of these developments are the manufacture of a precursor of insulin via rat genes inserted into bacteria, the production of human interferon (a potentially useful antiviral protein), and the production of a protein of the shape and size of human growth hormone via a piece of DNA containing the structural information for human growth hormone integrated into bacteria.
The useful proteins produced by such recombinant bacteria are typically trapped within the bacterial protoplasm and it is necessary to remove the cell wall surrounding the bacteria in order to free the useful protein. This has been done in the past by techniques such as sonification, freeze thawing or grinding techniques that physically destroy the bacterial cell wall. These techniques are both time consuming and non-specific and they are likely to interact with and cause denaturation and/or inactivation of the useful proteins.
The present invention provides processes and substances to significantly increase the yields from genetically engineered bacteria by enabling an efficient lysis of the bacterial cell wall. These novel immune proteins constitute a group herein termed P9. The P9 proteins also appear to be useful as pharmacological substances control certain bacterial infections.
P9 proteins are produced by injecting live bacteria into insect hemolymph and obtaining the P9 proteins from the induced hemolymph in a manner that does not destroy their bacteriolytic activity.
Prior to the present invention the hemolymphs of insects were studied in order to determine which substances were responsible for insect anti-bacterial activity, but little was known concerning the molecular basis for insect immunity to bacterial infection. As late as 1968 it was believed that the enzyme lysozyme fully accounted for all the phenomena of natural, acquired and passively acquired humoral immunity in insects. Mohrig, W. & Messner, B. (1968) Biol. Zentralbe. 87, 439-70. However, anti-bacterial factors with properties clearly different from those of lysozyme have also been reported. These were described as small, heat stable, acidic molecules of non-protein nature, Stephens, J. M. & Marshall, J. J. (1962) Can. J. Microbiol. 8, 719-725; Gingrich, R. E. (1964) J. Insect Physiol. 10, 179-94; Hink, W. F. & Briggs, J. D. (1968) J. Insect Physiol. 14, 1025-34; as small, basic proteins or co-factors, Kinoshita, T. & Inoue, K. (1977) Infect. Immun. 16, 32-36; Bakula, M. (1970) J. Insect Physiol. 16, 185-197; or as heat sensitive proteins, Natori, S. (1977) J. Insect Physiol. 23, 1169-1173.
The P9 proteins of this invention are clearly distinguished from known procaryotic proteins and eucaryotic proteins which are bactericidal for Escherichia coli. The only procaryotic proteins known to be bactericidal for E. coli are the colicins, Hardy, K. G. (1975) Bacteriological Reviews 39, 464-515, of which the only purified colicin which is known to be bacteriolytic is colicin M, Braun et al. (1974) Antimicrob. Agents Chemother. 5, 510-33. Colicin M, however, is clearly different from the P9 proteins of the present invention. The basic eucaryotic proteins present in polymorphonuclear leukocytes are bactericidal for E. coli but are distinguished from the present invention by their amino acid composition and molecular weight.
The subject matter of this invention relates generally to the subject matter of Hultmark et al., Eur. J. Biochem. 106, 7-16 (1980).